The COP9 signalosome (CSN) is an evolutionally conserved, essential multi-subunit protein complex critical for controlling diverse cellular and developmental processes in animals and plants. With 8 subunits (CSN1-CSN8), CSN functions as a deneddylase responsible for cleaving ubiquitin-like protein Nedd8 modification from neddylated substrates including cullin proteins, the key components of Cullin?RING ubiquitin E3 ligases (CRLs). CRLs represent the largest superfamily of multi-subunit E3s with more than 240 members, which orchestrate ~20% of protein degradation in the ubiquitin-proteasome system (UPS). The biological significance of CSN in eukaryotic biology is manifested by its function in controlling CRL activity and CRL-mediated protein degradation. CSN dysregulation has been implicated in many human diseases including cancers, and CSN inhibition has shown great potential for cancer therapy. A comprehensive understanding of how CSN works in cells is essential to uncovering molecular details underlying CSN biology and defining its role in human health and medicine. Recent structural analysis has revealed CSN architecture at medium resolution, and discovered that free CSN exists in an inactive state. It has been shown that CSN auto-inhibition can only be released through binding to neddylated cullins to induce substantial conformational changes required for CSN activation. Due to structural differences in numerous interchangeable substrate receptors (SRs), human CRLs have extremely high structural diversities within the family. Despite extensive structural studies, it remains unknown how CSN interacts with diverse structures of CRLs in cells to release its auto-inhibition and enable its function in regulating CRLs. We hypothesize that CSN might use dynamic topology to recognize and accommodate different CRLs. Due to limitations in traditional structural tools, novel approaches that can probe highly dynamic structures, compare a wide spectrum of conformational changes, as well as characterize in vivo structural topologies of large heterogeneous protein complexes, are needed to test this hypothesis. Recent work suggests that cross-linking mass spectrometry (XL-MS) is best suited for validating our hypothesis as it possesses all of the required capabilities. Therefore, we propose to develop and employ novel XL-MS technologies to effectively dissect the full-range of structural dynamics and conformational changes associated with CSN activation and function in vitro and in cells. To this end, our specific aims are 1) probing the structural dynamics of CSN using a combinatory XL-MS approach; 2) mapping conformational changes of CSN upon binding to CRLs in vitro and in vivo. The proposed project will not only help address important yet unresolved biological questions associated with CSN activation and function, but also propel quantitative XL-MS studies to a new level for studying dynamics and heterogeneous protein complexes in vitro and in vivo.